Over recent years cell culture has become a core technology in the life sciences. Cell culture is described in ‘Basic Cell Culture’ Oxford University Press (2002) Ed. J. M. Davis; and ‘Animal Cell Culture’ Oxford University Press (2000) Ed. John R. W. Masters; both of which are incorporated herein in their entirety by reference. Cell culture provides the basis for studying cellular processes such as the viability, phenotype, genotype, proliferation and differentiation of cells, and the formation of biological molecules, intermediates and products. It has also provided the means to study the regulation of these processes, from the genetic level—whether in isolation or in whole transgenic animals—down to the level of individual protein molecules. Notwithstanding its enormous contribution to the current state of biology, in many respects cell culture remains a developing discipline, albeit an unusually exciting science ultimately offering the possibility of genetic therapy and tissue engineering.
An important goal of cell culture is to be able to grow a wide variety of cells in vitro. The list of different cell types that can be grown in culture is extensive (see American Type Culture collection; European Collection of Cell Cultures; Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH), includes representatives of most cell types, and is continually increasing as more and more culture conditions are discovered. Despite the steady progress in the field, the method of determining suitable culture conditions for new cell types remains totally empirical: growth conditions are almost always discovered by trial and error. The choice of starting point will often be based on what was previously used by others for similar cells, or even what is currently being used in the laboratory for different cells. Many times these will simply be completely inadequate, and a process of trial and error must begin anew. Even when new culture conditions are successful, it is worthwhile remembering that adaptations of previous protocols will have introduced a historical bias to the experiment. For instance, much of the early tissue culture experiments made extensive use of fibroblasts, and to this date most standard cell culture conditions favour growth of cells derived from the mesoderm (fibroblasts, endothelium, myoblasts). The development of selective growth media for epithelial and other cell types based on these conditions was a challenge. For some of these cell types it is now known that serum—a normal component of many culture media for mesodermal cells—actually inhibits growth. One aspect of the invention described herein is a method for developing suitable culture conditions which allow for the viability, proliferation or growth, and retention of the phenotype of particular cell types.
Apart from conditions that favour cell proliferation, a particularly important step in modern tissue culture is to be able to control or direct the differentiation of cells towards a particular phenotype. As propagation of cell lines requires that the cell number increases, the vast majority of culture conditions have been developed to favour maximal cell proliferation. It is not surprising that these conditions are not conducive to cell differentiation, where cell growth is often limited or even abolished. The conditions which favour cell proliferation are generally low cell density, low Ca2+ concentration, and the presence of growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF). On the other hand, cytostasis and differentiation are promoted in conditions of high cell density, high Ca2+ concentration and the presence of differentiation inducers such as hormones (e.g. hydrocortisone), paracrine factors (e.g. IL-6, KGF, NGF), retinoids and even planar polar compounds such as dimethylsulphoxide (DMSO). Hence different conditions may be required for propagation and for differentiation of a particular cell line, and of course these respective conditions may differ between cells of different lineages. A second aspect of the invention described herein is a method for discovering suitable culture conditions which allow for the selective differentiation of cells.
Some common problems which are still encountered in cell culture are the limited lifespan of primary cell lines, the change of characteristics of cell lines with passage, and their transformation accompanied by loss of interesting cellular characteristics. These effects severely limit the utility of cultured cells for use in experiments or assays, for instance cell-based assays described below. Primary cells, i.e. cells freshly isolated from tissues, offer by far the most accurate cell culture models, as they behave in a way that broadly resembles their tissue of origin. Remarkably, a reliable method of culturing primary cells has still not been developed and consequently these cells exhibit a limited lifespan in vitro. This presents a serious technical limitation, for instance when attempting to amplify the primary culture, or when attempting to perform a longer-term experiment. A further problem associated with the use of primary cultures is that since they require constant fresh isolation, it can be hard to source primary material, particularly from humans and it is also difficult to obtain lines that behave consistently. A third aspect of the invention is therefore a method of culturing primary cells to obtain viable cultures with a prolonged lifespan.
If primary cultures are maintained in vitro for an extended period, they normally undergo a crisis in which the majority of cells perish, however the surviving cells are longer lived and can be cultured indefinitely. Most of these continuous cell lines are almost invariably poor representations of the cell as it is found in intact animal tissues. One reason for this lies in the fact that the process that allows the cells to become immortal also has an impact on the characteristics of the cell. For example, most established cell cultures have stopped expressing tissue-specific genes and instead only express housekeeping genes required for continuous growth in cell culture—as a result most such cell lines are more like each other than like the tissue from which they were originally sourced. For instance, most liver cell lines have stopped expressing the drug-metabolizing enzymes that would normally make them interesting tools for testing drug toxicity. A further aspect of the invention described herein is a method of culturing cells so that they provide more accurate models of tissues. This in turn would improve the reliability and predictive power of cell-based experiments and assays.
There is a need in the art for improved techniques for culturing cells, and methods for discovering and implementing such techniques for regulation of cellular processes such as growth, differentiation, metabolic activity, and phenotypic expression.